叶绿体型转昆虫抗冻蛋白基因烟草的耐寒性

根据已构建的大豆叶绿体表达载体pJY01，设计特异性引物，将昆虫抗冻蛋白基因MpAFP149插入此载体中构成叶绿体表达载体pJY01-MpAFP149，利用基因枪轰击法转化烟草，经壮观霉素筛选获得4株叶绿体型转抗冻蛋白基因烟草株系。PCR和PCR-Southern结果显示外源基因已整合至烟草叶绿体基因组中但同质化水平不高，RT-PCR结果也表明昆虫抗冻蛋白基因已发生了转录。将野生型烟草、叶绿体型转抗冻蛋白基因烟草及核转化T₁代转抗冻蛋白基因烟草(pCAMBIA1302- MpAFP149)于–1℃低温处理3 d，观察耐寒表型及测定相对电导率。结果表明, 叶绿体型转基因烟草的耐寒表型优于野生型烟草，但与核转化的T₁代转抗冻蛋白基因烟草无显著差异。处理3 d时，叶绿体型转基因烟草和T₁代转抗冻蛋白基因烟草的电导率分别为39.2%和38.2%，而野生型烟草已达73.7%。本实验获得的异质化转叶绿体抗冻蛋白基因烟草与转核基因烟草的耐寒力无差异。

Cold Tolerance of Transplastomic Tobacco Lines Carrying Insect Antifreeze Protein

The antifreeze protein gene MpAFP149 from desert insect Microdera punctipennis dzungarica was inserted into soybean chloroplast vector pJY01 to construct recombinant chloroplast vector pJY01-MpAFP149 by designing special primers. The plasmid was then transformed into tobacco by gene gun. Four transplastomic tobacco lines were obtained by spectinomycin screening. PCR and PCR-Southern analysis showed that the MpAFP149 gene was successfully integrated into the tobacco chloroplast genome, but the transgenicplants exhibited low homoplasmy. The result of RT-PCR also validated that MpAFP149 gene was transcribed at mRNA level. The antifreeze effect of transplastomic tobacco with insect antifreeze protein gene in its chloroplasts was evaluated by measuring the relative conductivity and comparing the phenotypes of different plants after cold treatment. Wild-type tobacco, transplastomic tobacco and T₁ generation of transgenic tobacco containing pCAMBIA1302-MpAFP149 in its nuclei were subjected to –1℃ for different days (0, 1, 2, and 3 d). The results showed that transgenic plants with insect antifreeze protein gene in chloroplasts or in nuclei performed better phenotype after cold treatment at –1℃ for three days and recovering at room temperature for five days than wild-type tobacco. After three days, the electrolyte leakage reached 73.7% for wild-type tobacco, 39.2% for chloroplast transgenic plants and 38.2% for nuclei transformed T₁ generation tobacco. There was no cold tolerance difference between nuclei transgenic tobacco and heterogeneous tansplastomic tobacco in our research.